By Madeline McCurry-Schmidt
Certain DNA sequences can get scrambled when cells divide, but the human body has built-in repair mechanisms to fix these mistakes. In a new study, researchers from The Scripps Research Institute (TSRI) have shown how a specific protein plays a role in removing unusual DNA sequences that can lead to cancer development.
Their findings, which focus on a repair protein called CtIP, were published recently online ahead of print by the journal Molecular Cell.
The new study by TSRI Associate Professor Xiaohua Wu and her colleagues brings scientists closer to understanding genome instability—and how to prevent it. “We’re really going through the mechanistic details to understand, step-by-step, how this works,” Wu said.
Watching DNA Repair in Action
To further investigate the role of CtIP and other factors important for DNA double-strand break repair, the team focused on regions of the genome called common fragile sites, where the nature of the DNA sequences allows the DNA to fold back on itself and form complex structures. When the DNA replication machinery encounters such complex structures, DNA can break. If not properly repaired, the broken DNA ends can fuse to other wrong chromosomal ends, causing genome instability.
This kind of genome instability and chromosome rearrangement is associated with cancer, and common fragile sites are “hotspots” for these rearrangements. “These genomic sequences, or regions, are present in all human beings, so it is important to understand how those sites are protected,” Wu said.
The team set up a DNA repair assay (test) based on the enhanced green fluorescent protein (EGFP) that originates from a kind of jellyfish and allows for cells to emit green signals. By watching for these green signals, the researchers monitored DNA repair within the common fragile sites.
First, the researchers inserted a DNA sequence, containing a common fragile site region from human DNA, into the EGFP gene, making it inactive and unable to express green signals. Because the common fragile site region consisted of repetitive pairs of the bases adenine and thymine (AT-repeats), it had the potential to fold back and form complex DNA structures. The scientists then used certain agents that cause replication stress to make the common fragile sites more susceptible to breaking.
The readout for repair was simple: If the cell was green, that meant there was a break made within the common fragile site and that break had been successfully repaired. Using this assay, the researchers tested the function of CtIP in the repair process and identified forms of CtIP that are defective for repair.
The team also used a technique called a nuclease assay to examine and characterize the enzymatic activity of CtIP involved in the DNA repair.
A New Role for CtIP
CtIP was previously known for its role in DNA end resection, a process where enzymes trim away the ends of a broken strand of DNA to facilitate repair of double-stranded breaks.
To Wu’s surprise, the results revealed that, in addition to aiding end resection, CtIP possesses an enzymatic activity that is specifically required for removing complex DNA structures from broken DNA ends that would block the repair process.
“It has to cleave this sort of weird structure to make the end normal, so the repair can go on,” Wu said.
She said the next step for the researchers is to expand beyond studying cells containing the engineered repair substrates to studying chromosomal regions in the human genome containing native common fragile sites and other regions prone to breakage. “What we really want to know is whether the same thing occurs at natural common fragile sites,” Wu said. “In this way, we will be able to better understand how the human genome is protected and its integrity is maintained to prevent tumors.”
In addition to Wu, other contributors to the paper, “CtIP Maintains Stability at Common Fragile Sites and Inverted Repeats by End Resection-Independent Endonuclease Activity,” were Hailong Wang, Yongjiang Li, Lan N. Truong, Patty Yi-Hwa Hwang, Jing He, Johnny Do, Michael Jeffrey Cho of TSRI; Linda Z. Shi and Michael W. Berns of Beckman Laser Institute, University of California, Irvine; Hongzhi Li and Binghui Shen of the Beckman Research Institute, City of Hope; Alejandro Negrete and Joseph Shiloach of the National Institutes of Health’s National Institute of Diabetes and Digestive and Kidney Diseases; and Longchuan Chen of the Veterans Affairs Medical Center, Long Beach, California. For more information, see http://www.cell.com/molecular-cell/abstract/S1097-2765(14)00323-2
The research was supported by the National Institutes of Health (grants CA102361, GM080677, CA140972, CA102361-07S1, CA140972-03S1, R01CA073764) and the Beckman Laser Institute Foundation.
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